The applicants previously showed that chromosome 6 suppresses human melanoma metastasis without affecting tumorigenicity, suggesting that a melanoma metastasis-suppressor gene is encoded on chromosome 6. Using differential display and subtractive hybridization, seven differentially expressed transcripts were identified and full-length cDNAs obtained. One clone with novel nucleotide and protein sequences, KiSS-1, was expressed only in nonmetastatic neo6/melanoma hybrids, normal melanocytes and RGP melanomas by Northern blot and by RT-PCR. The KiSS-1 gene mapped to the chromosome 1q32-q41 by FISH. Transfection of KiSS-1 suppressed human melanoma C8161 metastasis by 50-99%. Tumorigenicity was not affected in the transfectants. KiSS-1, therefore, met the criteria of a melanoma metastasis-suppressor gene. Three fundamental questions arise based upon the preliminary data--(1) Is KiSS-1 a universal melanoma metastasis-suppressor gene? (2) What regulates KiSS-1 expression? (3) How does KiSS-1 suppress metastasis? Specific Aim 1: will address the first question by transfection of KiSS-1 cDNA into other metastatic human melanoma cell lines and assessment of tumorigenicity and metastasis in athymic nude mice. Specific Aim 2: since KiSS-1 maps to chromosome 1, it is hypothesized that a gene(s) on chromosome 6 fragments, having already mapped a locus to 6q21-q23. For this aim, the applicants will continue using this strategy to further refine the locus to <5 Mb. Specific Aim 3: will test the hypothesis that KiSS-1 functions in a signaling pathway, mediating its metastasis-suppressor effects through interaction with an SH3-containing protein. This hypothesis is based upon the predicted amino acid sequence of the KiSS-1 protein. The most notable feature of the Mre18 kDa, largely-hydrophilic protein is a proline-rich domain with conserved PXP motif and flanking regions indicative of an SH3 domain ligand. In addition, KiSS-1 has multiple Ser, Thr and Tyr residues as well as a consensus PKC-alpha phosphorylation site, suggesting that it could also be phosphorylated. First, they will describe basic biochemical properties of KiSS-1 such as post-translational modification and cellular localization. Second, to directly test the hypothesis, we will prepare site-directed and deletion-mutant KiSS-1 and assess their abilities to suppress melanoma metastasis. Third, they will identify critical KiSS-1 protein/macromolecule interactions using plaque library screening an immune co-precipitation experiments.